DESCRIPTION: c-Abl is a ubiquitously expressed non-receptor tyrosine kinase localized to the nucleus and actin cytoskeleton whose kinase activity is regulated in the stress response to genotoxic insults. The kinase activity of c-Abl appears to be negatively regulated in vivo by a cellular inhibitor binding via the Abl SH3 domain, and Abl is activated to become leukemogenic by several mechanisms, including SH3 deletion and chromosomal translocations in human leukemias. A yeast two-hybrid screen for Abl SH3-binding proteins identified a gene called PAG, whose protein product (Pag) has many properties expected of a physiological inhibitor of c-Abl. When co-expressed with c-Abl, Pag associates with Abl in vivo and potently suppresses the tyrosine phosphorylation induced by c-Abl, dependent on the Abl SH3 and kinase domains. Pag also inhibits the kinase activity of c-Abl in an immune complex kinase assay. However, purified Pag is a poor inhibitor of purified Abl, suggesting that additional proteins might be required. Interestingly, Pag has been previously identified as a member of a novel family of proteins induced by oxidative stress which have antioxidant and cell cycle regulatory properties. The long-term goals of this application are to further our understanding of the regulation and physiological function of c-Abl, and to better understand the mechanism of activation of leukemogenic forms of Abl. In the first aim, the biochemical mechanism of inhibition of c-Abl by Pag will be determined. The location of the Abl SH3 and kinase domain interaction sites in Pag will be mapped. The inhibition of Abl observed in vivo will be reconstituted in vitro using Pag as an affinity reagent to isolate c-Abl from cell extracts. The identity of an additional cellular protein required for in vitro inhibition will be pursued biochemically, and an additional two-hybrid screen against Pag will be carried out. It will be tested whether Pag has antioxidant activity and whether oxidative stress activates c-Abl. In the second aim, it will be determined whether Pag is a physiological inhibitor of c-Abl in vivo. The subcellular localization of Pag and whether endogenous Pag and Abl co-associate will be determined, and whether this association is regulated by DNA damage or oxidative stress. The function of endogenous Pag will be inhibited by antisense and dominant-negative strategies, and the consequences for Abl activation and phenotypic responses determined. It will be tested if overexpression of Pag can inhibit transformation by SH3-containing oncogenic Abl. These experiments will yield important new information about the function and regulation of c-Abl and the mode of activation of leukemogenic forms of Abl, and identify new avenues for anti-leukemic therapies.